The goal of this project is to develop novel technology for targeted delivery of proteins to mitochondria. Defects in mitochondrial function are common in human diseases and are associated with significant mortality and/or birth defects. To overcome the limitations of viral vectors for delivering gene products to mitochondria inside of cells, we will use protein transduction domains (PTD) that cross cell membranes. We have recently shown that the Transactivator of Transcription (TAT) peptide from the human immunodeficiency virus can deliver proteins to mitochondria. We have further developed methods to localize these proteins to mitochondria by including a mitochondrial targeting sequence (MTS) in the fusion protein construct. The TAT-fusion protein crosses both cell and mitochondrial membranes and is localized because the MTS is recognized and cleaved leaving the fusion protein trapped in the mitochondria. This project will use a multidisciplinary team to test the hypothesis that TAT-fusion proteins can target biologically active proteins to mitochondria in the intact animal. We will: 1) Test the hypothesis that the TAT peptide can deliver an active mitochondrial protein in vitro. A fusion protein consisting of TAT and the mouse mitochondrial Trifunctional Protein (TFP) will be constructed and tested in cell culture. 2) Rescue the phenotype of an animal transgenic for loss of TFP. This will test the hypothesis that TFP can be delivered to tissues in vivo in adequate amounts to restore biological function. 3) Test !the hypothesis that TAT peptide can deliver proteins to different compartments in mitochondria. TAT will !be fused to an intermembranous space protein to show that the targeting sequence of the transduced protein remains operative in TAT fusion proteins. 4) Determine the effectiveness of novel PTDs for delivering proteins to mitochondria. Other PTDs will be tested to determine their effectiveness at crossing mitochondrial membranes. The significance of this work is the discovery and application of a novel therapy for patients with defects in mitochondrial function. The scientific importance of this work is the development of a non-viral platform technology that can be used to deliver gene products to mitochondria in multiple cell types and tissues that will be of value to scientists in multiple disciplines.